Comparison of the synthetic oligonucleotide gene probe and infant mouse bioassay for detection of enterotoxigenicEscherichia coli

A commercial DNA/DNA hybridisation kit for the detection ofEscherichia coli heat stable enterotoxin gene sequences was compared to the suckling mouse bioassay using 183 isolates ofEscherichia coli from clinical specimens. The gene probe assay had a specificity of 99 % and a sensitivity of 90.4 % compared to the infant mouse method. Using the colony blot method of preparing the bacterial DNA and a hybridisation temperature of 50 °C optimial results were obtained. The gene probe method is not affected by the incubation conditions of the test organisms. It is technically straightforward and can be applied to large numbers of specimens with fewer logistic difficulties than with the bioassy.     

Assessment of technique for rapid detection of Escherichia coli and Proteus species in urine by head-space gas-liquid chromatography.

A test depending on the production of ethanol by Escherichia coli from lactose and dimethyl disulfide by Proteus spp. from methionine in the early exponential phase of growth and the detection of these products by head-space gas-liquid chromatography has been applied to 75 specimens of urine selected to provide the most stringent trial of the test. The test was found to be rapid and reliable for the commonest findings in the microbiological examination of urine. In 3 to 4 h it detected "significant" numbers (greater than 10(5)/ml) of E. coli or of Proteus mirabilis or P. inconstans A, identified as Proteus spp., in 23 urines. It recorded the absence of infection from 32 urines containing borderline or "not significant" numbers of any organism. Significant numbers of other organisms in 13 urines were not mistaken for E. coli or Proteus spp. However, the test was less successful for some less common findings. Klebsiella ozenae in significant numbers in one urine was mistaken for E. coli. P. morganii in significant numbers in one urine was not detected. E. coli or P. mirabilis mixed with significant numbers of another organism were not detected in four out of five urines. The technique is simple and could be automated. It appears to merit more extensive trial in a hospital laboratory and further development to detect and correctly identify more species that cause urinary tract infections.     

Problems in interpretation of piperacillin susceptibility of TEM-1 producingEscherichia coli in the disk diffusion test

The susceptibility of 455Escherichia coli blood culture isolate to piperacillin was tested with the disk diffusion test. The presence of different beta-lactamase genes in these strains was also studied using DNA hybridization. Of the TEM beta-lactamase producing isolates, 64 % (61/95) were interpreted as intermediately susceptible to piperacillin. Because piperacillin is hydrolyzed by TEM-type beta-lactamases, we suggest that the intermediate susceptibility category should be reduced or omitted in testing piperacillin susceptibility ofEscherichia coli isolates.     

Detection of Epstein-Barr virus genomes in AIDS related lymphomas: sensitivity and specificity of in situ hybridisation compared with Southern blotting.

Eighteen cases of AIDS related, non-Hodgkin's lymphomas were examined for the presence of Epstein-Barr virus (EBV) genomes using in situ hybridisation with a 35S-labelled probe. The results were compared with those obtained independently by Southern blot analysis with a 32P-labelled probe of frozen tissue from the same tumours. Technically satisfactory results were obtained with both methods in 15 lymphomas. EBV DNA was detected in seven of 15 (47%) cases by in situ hybridisation and in eight of 15 (53%) cases by Southern blotting (including all the cases positive by in situ hybridisation). The results of EBV DNA detection by the two techniques were identical in 14 of 15 (93%) cases. In situ hybridisation gave no false positive results. This study shows that the sensitivity and specificity of in situ hybridisation for the detection of EBV genomes in AIDS related lymphomas approaches that of Southern blotting, even when using routinely processed archival, paraffin wax embedded material.     

Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system.

BACKGROUND: Amplification techniques such as PCR are becoming increasingly popular in the field of diagnosis of human cytomegalovirus (HCMV) also, thus substituting conventional techniques like the time consuming HCMV antigen or cell culture assays. Current PCR protocols however, are labor intensive, and moreover, the need for extensive postamplification manipulations increases the risk of false positive results due to contamination with amplified products. OBJECTIVES: to overcome these shortcomings, the new ultrarapid and semi-automated real-time LightCycler PCR-system (LC-PCR), which combines amplification and detection in a closed capillary system, was tested for its suitability in diagnosis of HCMV in urines. STUDY DESIGN: 73 urine samples from 64 newborns and infants suspected of having congenitally or postnatally acquired HCMV were tested with the LC-PCR and results were compared with those obtained in parallel with a conventional PCR-ELISA and the rapid shell vial assay for detection of HCMV early antigen (EA-assay). RESULTS: with these methods, 31 newborns/infants were found to be infected with HCMV. HCMV DNA was detected in 39 urines while the EA-assay was positive in 33 urines. All the EA positive samples were also positive for HCMV DNA. In the urines of the remaining 33 newborns (34 urine samples) neither HCMV DNA nor EA were detectable. The overall agreement of the two PCR tests was 100% while a 92% agreement was obtained between the PCR and the EA-assays. As the sensitivity of the three tests turned out to be quite similiar, the discrepancy observed in the positive rate between PCR and EA-assay is due to other factors which will be discussed in detail. However, while LC-PCR takes only about 2 h from sample preparation to result generation, the EA-assay, such as the conventional PCR-ELISA, needs 24-48 h. Furthermore, due to its capability to perform cycle-by-cycle monitoring, the LC instrument enables semi-quantitative analysis of HCMV viral-load. CONCLUSIONS: LC-PCR is a suitable new tool for routine analysis of HCMV in the urines of newborns and infants. Compared to the conventional PCR-ELISA a considerable increase in test rapidity and reliability is achieved without the need to sacrifice sensitivity.     

Direct detection of the human papovavirus BK in urine of bone marrow transplant recipients: comparison of DNA hybridization with ELISA

Urine specimens from bone marrow transplant (BMT) recipients and from controls were directly tested for BK virus (BKV) DNA sequences by dot hybridization and for BKV antigen by a double-antibody indirect ELISA. A total of 158 specimens from 55 BMT patients (57 collected prior to or at the time of transplantation and 101 in the posttransplant period) and single urines from 125 control subjects were examined by both methods. A molecularly cloned, 32P-labelled BKV probe was hybridized with urine sediments that were spotted directly on nitrocellulose filters and denatured in situ. BKV DNA sequences were detected in 1 (1.8%) pretransplant and 22 (21.8%) posttransplant urines of BMT patients, and in none of control urines. In ELISA of urine supernatants, BKV antigen was detected in 1 (1.8%) pretransplant and 21 (20.8%) posttransplant urines of BMT patients and in 1 (0.8%) of the control urines. The results of the two tests correlated as follows: 16 urines were positive and 253 urines negative by both methods; seven specimens were positive by DNA hybridization only and seven were positive by ELISA alone. Virus excretion in urine was demonstrated in 20 (36.4%) patients by DNA hybridization, in 19 (34.5%) patients by ELISA, in 15 (27.3%) patients by both methods, and in 24 (44%) patients by at least one of the two tests.     

Detection of human polyomavirus DNA in urine specimens by hybridot assay

Summary A hybridot assay method using a labelled BK virus probe has been used to detect the presence of human polyomavirus DNA in 81 urine specimens from 61 patients, most of whom were immuno-compromised to some degree. Twenty-eight urines from 23 patients had detectable DNA. The results have been compared to virus isolation and electron microscopy on the same specimens. In 90 per cent a comparable result was obtained by at least one of the other two tests and in the 73 specimens on which all 3 tests were performed there was 80 per cent agreement between all. In 6 cases the hybridot assay was more sensitive in detecting infection.With 1 Figure     

A comparative study of specific gene probes and standard bioassays to identify diarrhoeagenic Escherichia coli in paediatric patients with diarrhoea in Bangladesh.

We compared the usefulness of gene probes with standard bioassays to identify diarrhoeagenic Escherichia coli amongst isolates from Bangladeshi children under 1 year of age with diarrhoea. E. coli isolates were analysed with specific gene probes for localised adhesiveness (LA), diffuse adhesiveness (DA), heat-labile toxin (LT), heat-stable toxin (ST), Shiga-like toxins (SLT I and SLT II), and enteroinvasiveness, and in bioassays for production of enterotoxins and cytotoxins, and for cell adherence. With 1136 isolates from 387 patients, there was general agreement between the two assay methods. When there was disparity, gene-probe-positive isolates gave negative results in the corresponding bioassay. In the HeLa cell adherence assay, 94% of the LA probe-positive isolates and 91.6% of the DA probe-positive isolates gave positive bioassay results for LA and DA respectively. Thirty-six of 39 LT probe-positive isolates and 73 of 86 ST probe-positive isolates gave positive results in the bioassays. Of 28 isolates that gave negative results in the suckling mouse assay but were initially positive with the probe for ST, 15 were later found to hybridize with the cloning vector for the ST probe. Addition of denatured vector DNA at a concentration of 10 micrograms/ml in the hybridisation solution eliminated these false positive results. None of the other probe-positive isolates hybridised with any of the cloning vectors used. The DNA hybridisation assay appeared to be a convenient alternative to bioassays for screening large numbers of isolates in epidemiological investigation.     

Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE

To determine whether confirmatory tests for extended-spectrum beta-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was >/=2 micro g/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by >/=3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by >/=5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla(TEM), bla(SHV), and bla(OXA), and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one beta-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 micro g/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 micro g/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC beta-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to >/=8 micro g/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.     

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis

A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.     

Detection of Campylobacter pylori in stomach tissue by DNA in situ hybridisation.

A non-radioactive DNA in situ hybridisation (DISH) protocol was developed. It requires the use of biotinylated Campylobacter pylori DNA as the probe to detect C pylori DNA in routinely embedded stomach biopsy specimens. In sequential tissue samples from a 58 year old woman with recurrent chronic active gastritis the C pylori probe hybridised with bacteria whenever they were histologically visible. When no bacteria were visible histologically, hybridisation was negative with one exception. In a single biopsy specimen without visible C pylori, hybridisation occurred with the surface of the antrum epithelium, while control hybridisation with a heterologous probe remained negative. From a parallel biopsy specimen taken at the same time the C pylori culture was positive. It is concluded that DISH, although more laborious than routine staining techniques, may be more sensitive in that it detects bacteria very easily, even when sections are not counterstained or when they are present in low numbers, and that bacteria which do hybridise are unequivocally identified as C pylori and not Campylobacter-like organisms.     

Prevalence of extended-spectrum β-lactamases in isolates of the Enterobacter cloacae complex from German hospitals

In 2002, 119 isolates of the Enterobacter cloacae complex were collected randomly from 11 German laboratories nationwide. Antibiotic susceptibilities were tested by disk-diffusion tests according to CLSI guidelines, and MICs were determined using Etests. PCRs were performed to amplify all TEM and SHV, and most CTX-M and OXA beta-lactamase genes. PCR products were sequenced to identify the precise extended spectrum beta-lactamase (ESBL) types. Isoelectric focusing (IEF) and PM/PML Etests were used to confirm production of the respective ESBLs. According to susceptibility tests and CLSI criteria, 49 (40%) isolates were resistant to extended-spectrum cephalosporins. Seven (5.8%) isolates were positive in at least one of the PCR assays. Sequencing identified production of TEM-1 beta-lactamase genes by three (2.9%) isolates, and ESBL genes of the CTX-M and SHV beta-lactamase families by five (4.2%) isolates. IEF confirmed the production of beta-lactamases in the expected pI ranges of the respective ESBLs, and four of the five ESBL-producers were detected using the PM/PML Etest. All ESBL-producing isolates showed co-resistance to sulphonamides.     

Comparison of five assays for the heat-labile enterotoxin of Escherichia coli

Enzyme-linked immunosorbent assay (ELISA), DNA-DNA hybridisation, Vero cell assay, the Biken test and a new membrane-filter method were compared in the detection of heat-labile enterotoxin (LT) of Escherichia coli. Six subcultures of each of 50 strains of E. coli from the Biken collection were evaluated "blind" in the laboratory. The combined results of the most reproducible tests (ELISA and DNA-DNA hybridisation) were used to calculate the sensitivity and specificity of the other assays. The Vero-cell assay had a high sensitivity (98%) but a lower specificity (91%). The Biken and membrane-filter assays had sensitivities of 58-71% and 77-84% respectively, depending on the type of antiserum used. Only one false positive result was obtained with the Biken test; specificity of the membrane-filter assay was 94-95%. The membrane-filter assay, with anti-cholera toxin, is specific and reasonably sensitive. It has particular advantages over DNA-DNA hybridisation and the Biken test, and it may prove suitable for screening large numbers of E. coli isolates in epidemiological studies in developing countries.     

DNA fingerprints of Helicobacter pylori before and after treatment with omeprazole.

AIMS: To test whether a hypoacidic environment may potentially "stress" Helicobacter pylori DNA, encouraging the emergence of strain variation. METHODS: This hypothesis was tested by inducing prolonged hypoacidity with omeprazole, a potent antisecretory drug. The genomic DNA of H pylori was studied by electrophoretic separation of restriction endonuclease fragments followed by rRNA gene hybridisation in seven patients infected with H pylori before and after treatment with omeprazole 20-40 mg daily for six to eight weeks. DNA was isolated and purified using the guanidium thiocyanate reagent method. DNA samples were digested with Hae III, electrophoresed, vacublotted, and hybridised using a biotinylated cDNA probe prepared from 16S and 23S rRNA from H pylori NCTC 11638. Isolates were compared using their ribopatterns (DNA fingerprints). RESULTS: A total of 26 isolates were obtained; all DNA isolates were cut by Hae III, which was the enzyme that gave the best resolved hybridisation patterns for analysis. No two patients harboured the same strain. The isolates from two patients showed evidence of subtypic variation; one patient had two distinct strains and four patients had their own indistinguishable strains before and after treatment with omeprazole. For each patient, the paired ribopatterns of H pylori DNA were not affected by treatment with omeprazole for six to eight weeks. CONCLUSION: The H pylori genome is relatively stable when exposed to the conditions of prolonged hypoacidity that result from treatment with omeprazole.     

Comparison of polyethylene glycol precipitation and ultracentrifugation for recovery of cytomegalovirus from urine prior to detection of DNA by dot-blot hybridisation

Polyethylene glycol (PEG) precipitation provided a useful alternative to ultracentrifugation for recovering cytomegalovirus (CMV) from clinical specimens prior to DNA hybridisation studies. The conditions for use of PEG are described. In a study of 61 urine samples from patients suspected of CMV infection 18 yielded a positive DNA hybridisation result. Fifteen were positive after both concentration procedures, two after PEG precipitation only and one after ultracentrifugation only. The DNA hybridisation results are discussed in the light of the results of virus isolation by cell culture.     

Evaluation of Gram-stain screen and Micro-ID methods for direct identification ofEnterobacteriaceae from urines

A rapid method of urine screening and enterobacterial identification was evaluated. Results indicated that an average of 13.5 bacteria/oil immersion field (threshold value 1) was observed in unsedimented urine of patients with significant bacteriuria, with an average of < 1 bacterium/field in urines of patients without significant bacteriuria. In centrifuged urines, numbers of bacteria divided by amount of urine sedimented yielded similar results. Of 1758 urines studied, 136 yielded 10⁵ bacteria/ml, and 58 > 10⁴ but < 10⁵ bacteria/ml, by conventional techniques. Gram-screening of unsedimented specimens gave sensitivity rates of 94.1%, specificity of 97.7%, and predictive positive and negative values of 78.5%, 99.5%, respectively; similar values were obtained with sedimented urines. Sensitivity rates of both screening methods for the 58 urines with > 10⁴ but < 10⁵ bacteria/ml were 9.0%, 10.0%, respectively. Total correct enteric identification in 113 urines with positive screens and significant bacteriuria ( 10⁵/ml) was 82.3% and 90.3% with direct saline and broth Micro-ID methods, respectively. In 99 urines yielding pure or predominantly pure growth of 1 species ofEnterobacteriaceae identification by direct saline and broth Micro-ID corresponded with isolated colony identification in 85.9%, 94.9% of cases, respectively. Gram-stain screening (together with back-up conventional plating in certain patient categories) and enterobacterial identification by direct broth Micro-ID, of urines with pure stains suggestive of 10⁵ Gram-negative rods/ml has been shown to be useful in laboratories without automated equipment for urine screening.Presented in part at the American Society for Microbiology Annual Meeting, Dallas, Texas, March 1981     

Widespread presence of cytomegalovirus DNA in tissues of healthy trauma victims.

AIMS: To determine the localisation of human cytomegalovirus (CMV) DNA in abdominal aorta, spleen, and transplantable organs, such as kidney, pancreas, and liver, obtained from healthy individuals; to characterise the cell type(s) in these tissues that serve as a reservoir for latent CMV. METHODS: CMV DNA was detected by dot blot DNA hybridisation and in situ DNA hybridisation with a probe for CMV major immediate early sequences (UL123) and nested PCR with primers derived from the CMV major immediate early (IE) gene exon 4 (UL123ex4). Samples of liver, abdominal aorta, spleen, kidney, and pancreas were obtained at necropsy or from donor kidneys from healthy subjects. RESULTS: CMV DNA was detected in most tissue samples using dot blot hybridisation and nested PCR. In situ hybridisation demonstrated that, in addition to smooth muscle cells in the arterial wall, hepatocytes, tubular and glomerular kidney cells, splenic red pulp cells, and pancreatic acinar cells also harboured CMV DNA. CMV DNA was detected in seropositive and in some seronegative subjects. CONCLUSION: CMV DNA is widely distributed in organs of healthy subjects. CMV DNA was found in various cell types in several organs, suggesting that during latency, CMV DNA is present thoughout the body.     

HIV-1 DNA in fibroblast cultures infected with urine from HIV-seropositive cytomegalovirus (CMV) excretors

Summary Interactions between HIV-1 and CMV may be important in the pathogenesis of AIDS. We have studied whether active CMV infection alters the cell tropism of HIV-1 in dually-infected individuals. Urines from HIV-seropositive individuals excreting CMV were compared to urines from CMV non-excretors. Sixty-six urines from HIV-seropositive individuals were tested. Infectious HIV-1 was not detected in any of the concentrated urines tested. The urines were filtered, concentrated, DNase-treated and cultured on HIV-1 non-permissive human forestin fibroblasts. HIV-1 DNA was detected by PCR withpol gene primers in 5 of 39 MRHF cell cultures inoculated with CMV culture positive urine (p=0.037). HIV-1 DNA was not detected by PCR in uninfected fibroblasts, in fibroblasts inoculated with CMV uninfected urine from 27 HIV-seropositive patients or in fibroblasts cultured with 9 CMV culture positive urines from 16 HIV-seronegative renal transplant recipients. Supernatant fluid from an HIV-1 PCR-positive culture was passaged onto another fibroblast monolayer, and these cells were negative for HIV-1 DNA. Direct inoculation of fibroblasts with HIV-1 did not yield evidence of infection by PCR. CMV infection may facilitate HIV-1 DNA entry into ordinarily non-permissive cells.     

Increase in incidence of resistance to ampicillin, chloramphenicol and trimethoprim in clinical isolates of Salmonella serotype Typhimurium with investigation of molecular epidemiology and mechanisms of resistance.

Antimicrobial resistance patterns of Salmonella serotype Typhimurium isolates obtained during the period 1987-1994 were examined and the molecular epidemiology and the mechanisms of resistance to ampicillin, chloramphenicol and trimethoprim were investigated in 24 strains isolated during 1994. Resistance to ampicillin increased from 18% to 78%, to chloramphenicol from 15% to 78%, to tetracycline from 53% to 89% and to co-trimoxazole from 3% to 37%, whereas resistance to norfloxacin remained at 0%. Of Salmonella serotype Typhimurium strains isolated during 1994, all ampicillin-resistant strains had an MIC > 256 mg/L, except one strain in which the MIC was 64 mg/L. Twelve strains (52%) had a TEM-type beta-lactamase, nine (39%) a CARB-type beta-lactamase and two strains (8%) had an OXA-type beta-lactamase. Chloramphenicol acetyl-transferase activity was detected in only nine (47%) of 19 chloramphenicol resistant strains, whereas all eight trimethoprim-resistant strains produced a dihydrofolate reductase type Ia enzyme. Three different epidemiological groups were defined by either low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis or repetitive extragenic palindromic-PCR. The latter technique provided an alternative, rapid and powerful genotyping method for S. Typhimurium. Although quinolones provide a good therapeutic alternative, the multiresistance of S. Typhimurium is of public health concern and it is important to continue surveillance of resistance levels and their mechanisms.     

Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells.

A fast and sensitive method for detecting biotinylated deoxyribonucleic acid (DNA) probes was used for sex determination of cells and tissues by in situ hybridisation of a probe "specific" for the Y chromosome (pHY 2.1). Within 24 hours this procedure visualizes the Y chromosome in fetal and adult cells and tissue, without background noise. This procedure should facilitate antenatal determination of sex on small numbers of uncultured cells. The sensitivity of the procedure also permits the chromosomal assignment of genes present in low copy number.